Project Summary The ocular lens is responsible for fine focusing of light onto the retina. Age-related changes in lens mechanics are linked to presbyopia, a reduction in the lens' ability to change shape during focusing (accommodation). While mouse lenses do not accommodate, they do show age-dependent stiffening like primates and other mammals, and provide a genetic model system to elucidate cytoskeletal regulation of cellular architecture, transparency and mechanics during aging. The lens consists of a bulk of elongated, hexagonally packed fiber cells, which undergo organelle loss and compaction to minimize light scattering, with formation of complex interlocking membrane protrusions at cell-cell interfaces. Lens fibers contain a spectrin-actin network with two F-actin-stabilizing proteins, tropomodulin1 (Tmod1) and tropomyosin (?TM). Tmod1-/- mouse lenses have reduced stiffness and abnormal fiber cell interlocking domains, and ?TM-/- lenses have reduced stiffness with progressive anterior cataracts. The lens also contains nonmuscle myosin IIA (NMIIA), and human lenses with mutations in the NMIIA heavy chain, termed MYH9-related disorders (MYH9-RD), develop cataracts. This proposal will test the hypotheses that F-actin stability mediated by Tmod1 and ?TM, and tensile forces mediated by actomyosin contractility, function to confer distinct fiber cell architectures during differentiation and maturation, thereby determining lens optical and mechanical properties that vary radially with fiber cell maturation, and temporally with aging. Aim 1 will elucidate the relationship between lens mechanics, fiber cell architecture and F-actin networks in wild-type mouse lenses as a function of aging. Detailed lens biomechanical properties will be measured with an Instron instrument, and responses of fiber cell shapes and F-actin networks to compression (and release) evaluated by immunostaining, confocal microscopy and scanning electron microscopy across the lens radius, and biochemistry of whole lenses. We predict that age- dependent cytoskeletal reorganization at the fiber-cell level contributes to age-dependent stiffening and loss of resilience of the mouse lens at the whole-organ level. Aim 2 will test the role of F-actin stability by analyzing lenses from Tmod1-/-, ?TM-/- or Tmod1/?TM double knockout mice. We predict that Tmod1 and ?TM cooperate to stabilize the spectrin-actin network in interlocking domains of mature fibers, thereby regulating age-dependent lens transparency, mechanical stiffness and resilience. Aim 3 will test the role of actomyosin contractility by analyzing lenses from transgenic knock-in mouse models with mutations in the NMIIA motor domain (R702C) or rod domain (D1424N, E1841K) that phenocopy human MYH9-RD. We predict that loss of F-actin stability, or impaired actomyosin contractility, result in distinct cytoskeletal and cellular reorganizations at the fiber-cell level that contribute to age-dependent stiffening and loss of resilience of the mouse lens at the whole-organ level. This project is the first to use a multidisciplinary, integrative approach to link age-dependent changes in lens biomechanical properties to alterations in fiber cell architecture and cytoskeletal organization.